Fig. 5.

TGM2 upregulation results in the accumulation of HIF-1a under normoxic conditions promoting EMT in HCC cells: PGK1, LDHA, ENOG, and HIF-1a expression levels increased in HCC cells (MHCC97H and HepG2) co-cultured with LX2 cells (a) and increased in MHCC97H-TGM2 OE and HepG2-TGM2 OE cells compared with counterpart cells (western blotting). b, c Heightened HIF-1a expression in tumour cells following combined injection of MHCC97H and LX2 cells into nude mice, and following injection of MHCC97H-TGM2 OE cells, compared with counterpart cells injection (Immunohistochemistry, scale bar, 50 μm); d Heightened HIF-1a expression in both cytoplasm and nucleus of MHCC97H-TGM2 OE and HepG2-TGM2 OE cells, compared with MHCC97H-Mock and HepG2-Mock cells, respectively (western blotting); e Higher E-cadherin and lower Vimentin expression levels in HCC cells after plasmid-mediated HIF-1a knockdown co-culturing with LX2 cells (MHCC97H-Mock’-shHIF-1a-Co and HepG2-Mock’-shHIF-1a-Co), compared with two HCC cell lines subjected to plasmid control co-culturing with LX2 cells (MHCC97H-Mock’-Co and HepG2-Mock’-Co), respectively (western blotting). Note higher E-cadherin and lower Vimentin expression levels in MHCC97H-TGM2 OE and HepG2-TGM2 OE cells after plasmid-mediated HIF-1a knockdown (MHCC97H-TGM2 OE-shHIF-1a and HepG2-TGM2 OE- shHIF-1a), relative to two HCC plasmid control cell lines (MHCC97H-TGM2 OE and HepG2-TGM2 OE), respectively (western blotting); f Dot plots showing no significant difference in oxygen concentration of culture medium in HCC cells co-cultured with LX2 cells (MHCC97H-Co and HepG2-Co) and HCC cells alone or in HCC cells showing TGM2 upregulation (MHCC97H-TGM2 OE and HepG2-TGM2 OE) and control HCC cells (MHCC97H-Mock and HepG2-Mock), as registered by dissolved oxygen metres; g No significant difference in HIF-1a gene expression in HCC cells co-cultured with LX2 cells and in HCC cells alone, or in HCC cells showing TGM2 upregulation and control HCC cells (quantitative real-time polymerase chain reaction); h Higher hydroxy-HIF-1a and lower VHL expression levels in MHCC97H-TGM2 OE and HepG2-TGM2 OE cells, compared with MHCC97H-Mock and HepG2-Mock cells, respectively (western blotting); i Immunoprecipitation of MHCC97H and HepG2 cell lysates, with or without TGM2 upregulation, using anti-TGM2 antibody (confirmatory western blot using anti-TGM2 and anti-VHL antibodies); and j TGM2 binding and polymerisation of VHL result in degradative VHL depletion, causing HIF-1a to accumulate. Experiments were repeated three times in triplicate